Methods, compositions and apparatus for delivering heterologous molecules to cells

ABSTRACT

A method for transferring a macromolecular complex to muscle cells by exsanguinating a region of the subject&#39;s microvasculature and delivering the complex to this region under high hydrostatic pressure is described. A balloon catheter having a balloon that extends substantially the full length of the cannula that is inserted into the subject is provided for use in the systemic delivery of macromolecular complex.

BACKGROUND OF THE INVENTION

The invention relates generally to the field of gene therapy.

A variety of methods have been described in the literature as beinguseful for delivering a desired molecule into a target cell. Thecombined use of 1) complete vascular isolation using tourniquets andproximal arterio-venous cannulation, 2) systemic heparinization toprevent thrombosis, 3) peripheral vasodilation to optimize perfusion ofmuscle capillaries, and 4) histamine to produce physical gaps betweenadjacent endothelial cells has achieved widespread, homogeneous,vector-independent gene transfer to muscles of entire extremities. Thesestudies began with marker genes and were then applied to several diseasemodels in larger rodents and dogs. However, the four essentialcomponents of the protocol listed above have stymied clinicaltranslation because of the inherent risk of hemorrhage, hypotension, andpulmonary dysfunction.

For example, U.S. Pat. No. 6,177,403 describes a kit for delivering amacromolecular assembly to the extravascular tissue of an animal. Thiskit involves the use of a vascular permeability-enhancing agent and avasodilating agent. However, such agents can be associated withundesirable side effects, including short-term toxicity, which minimizesthe usefulness of such methods.

Systems for pressure mediated selective delivery of therapeuticsubstances to specific areas of organs and cannula useful therein havebeen described. See, e.g., WO 99/59666, Nov. 25, 1999. However, thesemethods and devices avoid systemic delivery of therapeutic substances.

Further, studies in large animal models have revealed a trade-offbetween the efficiency of gene transfer using known methods and theinherent safety of the required pharmacological interventions.

What are needed are methods that facilitate delivery of target moleculesto the desired host cell while minimizing side effects.

SUMMARY OF THE INVENTION

The present invention provides a method of transferring a macromolecularcomplex from the vascular space to the interstitium in a subject in theabsence of permeability enhancing agents by isolating a region of thesubject's microvasculature and partially or completely exsanguinatingthat region. Thereafter, the complex is delivered to the exsanguinatedregion under rapidly applied high hydrostatic pressure. Also provided isa balloon catheter for systemic delivery of the macromolecular complexof the invention and kits useful for performing the method of theinvention.

The present invention provides the first protocol for somatic genetransfer to muscle that achieves scale-independent, limb-widetransduction of nearly 100% of fibers, while avoiding the risks ofhistamine, papaverine, heparin, and arterial access.

Other aspects and advantages of the present invention will be readilyapparent from the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic side elevational view of an internally occlusiveaortic balloon catheter according to the present invention with acannula shaft configured for retrograde deployment via femoral arteryaccess;

FIG. 2 is a schematic side elevational view of an internally occlusiveaortic balloon catheter according to the present invention with acannula shaft configured for retrograde deployment via femoral arteryaccess, and showing compartments in the balloon, each compartmentcontaining separate fluid ports;

FIG. 3 is a schematic anterior view of a patient, illustrating placementof the aortic balloon catheter of FIG. 1;

FIG. 4 is a schematic anterior view of a patient, illustrating placementof a segmented balloon catheter in the vena cavae;

FIG. 5 is a schematic posterior view of the heart of the patient in FIG.4; and

FIG. 6 is a schematic view, similar to FIG. 5, showing details of theballoon catheter placement.

FIG. 7 illustrates an open chest during a cardiopulmonary procedure.Illustrated are all important components of the cardiac isolation methodof the invention, including the aortic cross-clamp 11, superior venacava (SVC) cannula 13 and inferior vena cava (IVC) cannula 15, SVC 21and IVC 23 snares, pulmonary cross-clamp 25, cardiac arterial in-flow31, right ventricular (RV) 33 and left ventricular (LV) vent catheters35, and the coronary sinus catheter 29.

DETAILED DESCRIPTION OF THE INVENTION

The inventors provide a method by which rapid, mechanical distention ofthe venular endothelium by afferent infusion from a distal site safelyfacilitates macromolecular transport from the vascular space to themuscle interstitium (e.g., skeletal muscle). Pressurized infusionthrough a large-bore catheter in a distal, superficial vein according tothe invention results in uniform, scale- and vector-independenttransduction of myofibers in anatomic domains isolated from the centralcirculation by tourniquet. This approach is rapid, minimally invasive,and avoids pharmacological interference with cardiovascular homeostasis.Accordingly, the present invention provides uniform gene transfer to themuscle fibers of an entire extremity in a mammal larger than a rodent,which is applicable to large mammals, including humans.

In one aspect, the invention provides a method of transferring aheterologous macromolecular complex from the vascular space to theinterstitium surrounding muscle cells in a subject in the absence ofpermeability enhancing agents by delivering the complex to a subject'smicrovasculature under high hydrostatic pressure. When performedsystemically, this method is performed with the patient under totalcirculatory arrest and under hypothermic conditions.

Exsanguination followed by high pressure delivery of a heterologousmolecule to the interstitial space between the epithelial cells of themicrovasculature and into surrounding muscle cells facilitates entry ofthe molecule into the interstitial space. Further, subjecting thepatient to hypothermia, according to one aspect of the inventioninvolving isolation of the central and peripheral circulation, enhancessafe entry of the molecule.

Without wishing to be bound by the mechanism of the invention, theinventors theorize that the reasons for the success of the method of theinvention in obtaining efficient delivery of macromolecules to largeranimals are based upon differences in the anatomy of mice, which aretypically used in initial gene therapy studies and larger animals,including humans. More particularly, the inventors have noted that theveins of larger mammals, including humans contain one-way valves inlarge veins and in vessels down to as small as about 1 mm; whereas theseperipheral valves do not exist in mice. The inventors theorize that thepresence of these valves contributes to preventing efficient genetransfer in larger animals. Additionally, the inventors have noted thatthe basal laminate of the vessels becomes progressively thicker inlarger animals as from the top of the animal (e.g., head, neck) to thebottom of the animal as these vessels are subject to a higher pressuregradient. This thickening of the basal laminate is not present in mice,which have even been observed to have a “leaky” vasculature. In view ofthis theory, the inventors have termed the method of the inventionATVRx, Afferent TransVenular Retrograde eXtravasation. These anatomicaldifferences between a small mammal such as the mouse and larger mammalswere not previously recognized as significant with respect to deliveryof macromolecules to muscle cells prior to the present invention.

The term “heterologous” includes, among other things, molecules that arenot natively found in combination with the material with which they arebeing associated. For example, a heterologous molecule is not found in atarget cell in the form in which it is delivered to the cell. As anotherexample, heterologous refers to molecules, including nucleic acidsequences, which are derived from the same source but are nativelynon-contiguous, or molecules that are derived from different sources.This definition is not a limitation on the present invention.

The method of the invention is well suited for avoiding an immuneresponse, and particularly, a response from circulating antibodies. Themethod of the invention is performed following isolation and at leastpartial resanguination of a region of the vasculature, followed bydelivery of the macromolecular complex. The absence of circulating bloodin the area to which the macromolecular complex is infused minimizes therisk of inducing an immune response and clearance of the macromolecularcomplex by neutralizing antibodies. Thus, in one embodiment, the methodof the invention permits administration, or readministration, of amacromolecular complex to which the patient has preexisting circulatingantibodies. In some embodiments, resanguination of the targeted portionof the microvasculature follows flushing of residual macromolecularcomplex (i.e., macromolecular complex not taken up by the extravasculartissue, e.g., muscle cells) from the area following infusion. Thisflushing step can be performed by washing the area with saline solutionthat contains no complex. Thus, the method of the invention minimizesthe exposure of the subject to the complex, thereby reducing the risk ofan immune reaction. Further, the method of the invention minimizes, oreliminates, exposure of other non-targeted areas of the body to thecomplex. For example, when the area targeted is a limb, isolation of thelimb minimizes or completely eliminates exposure of the liver or lung tothe complex.

Advantageously, the method of the invention also avoids unwantedelements of the blood, e.g., cells, platelets, and tissue-reactiveplasma components, from contacting the macromolecular complex. Thus, themethod of the invention also avoids activation of various clottingfactors and other factors that could interfere with the transfer of themacromolecular complex.

The invention further provides compositions and devices useful forperforming this method, as well as other functions that will be apparentto those of skill in the art given the guidance provided in thisapplication.

As used herein, the term muscle cells include both skeletal muscle cellsand smooth muscle cells. In one embodiment, the muscle cells are cardiacmuscle. However, other muscle cells can readily be targeted.

As used herein, the term “high hydrostatic pressure” generally refers toa pressure in the range of 50 mm Hg to 500 mm Hg. Suitable pressureswithin this range, e.g., 75 mm Hg, 100 mm Hg, 150 mm Hg, 200 mm Hg, 250mm Hg, 300 mm Hg, 350 mm Hg, 400 mm Hg, or 450 mm Hg, or others withinor outside this range may be readily selected. Some of the valuesprovided herein are measured in torr, which at 0° C. is equivalent to mmHg. High hydrostatic pressure is applied according to the invention by alow resistance (large bore) catheter or cannula in either a vein orartery, or by other methods that will be readily apparent to one ofskill in the art.

Typically, the high hydrostatic pressure described herein is rapidlyapplied by way of at least one low resistance catheter or cannula ineither a vein or an artery. Currently, in a preferred embodiment, thecatheter or cannula is applied in a vein.

I. Macromolecular Complex

As used herein, the term “macromolecular complex” encompasses anybiologically useful moiety that can be transferred into targeted cells(e.g., muscle cells). Examples of suitable macromolecular complexesinclude vectors composed of nucleic acids, including RNA and DNAmolecules, dominant negative mutants, an enzyme, a protein, peptide, ornon-proteinaceous molecule, which may include small molecules or otherchemical moieties.

Desirable RNA molecules include tRNA, dsRNA, ribosomal RNA, catalyticRNAs, siRNA, small hairpin RNA, trans-splicing RNA, and antisense RNAs.These RNA molecules can be delivered in the form of a transgene carriedby a vector or by other suitable means.

The macromolecular complexes of the invention are not limited by size,but rather encompass molecules that, due to their large size, are notable to enter the cell on their own as well as molecules that can infector transfect cells without the application of the present method.

A. Vectors

A vector includes plasmids, episomes, cosmids, viral vectors, phage,“naked DNA”, any of which desirably contains a transgene under thecontrol of regulatory sequences that direct expression thereof in atarget cell.

The transgene is a nucleic acid sequence, heterologous to the vectorsequences flanking the transgene, which encodes a polypeptide, protein,or other product, of interest. The nucleic acid coding sequence isoperatively linked to regulatory components in a manner that permitstransgene transcription, translation, and/or expression in a host cell.Suitably, these transgenes may also carry a desired RNA molecule, asdescribed herein.

In one embodiment, the macromolecular complex comprises a viral vector.Examples of suitable viral vectors include, without limitation,adenoviruses, picornavirus, adeno-associated viruses, retroviruses,baculoviruses, and lentiviruses, among others. For example, amacromolecular complex can be an adenoviral vector comprising a humanminidystrophin gene [Ragot et al, 1993, Nature 361:647-650) orpAdDeltaRSV, which is a modified plasmid containing a full-lengthdystrophin cDNA [Koening et al, 1998, Cell 53:219-228] with a backboneof a pBSA-2 vector with an RSV promoter operably linked to thedystrophin cDNA, and containing adenoviral 5′ and 3′ ITRs flanking thepromoter-dystrophin cDNA. Currently, adeno-associated viruses (AAV) areconsidered particularly well suited for delivery to muscle. Typically, arecombinant AAV is composed of, at a minimum, a transgene and itsregulatory sequences, and 5′ and 3′ AAV inverted terminal repeats(ITRs), all packaged in a capsid composed at least in part of proteinsencoded by an AAV “cap” gene. In one desirable embodiment, the ITRs ofAAV serotype 2 are used. However, ITRs from other suitable serotypes maybe selected. However, the invention is not limited to use of viralvectors or, when viral vectors are selected, to use of rAAV.

B. Transgene

When present in a macromolecular complex as defined herein, a transgeneis selected with regard to the biological effect desired.

One example of a useful RNA sequence is a sequence which inhibits orextinguishes expression of a targeted nucleic acid sequence in thetreated animal. Typically, suitable target sequences include oncologictargets and viral diseases. See, for examples of such targets, theoncologic targets and viruses identified below in the section relatingto immunogens.

Another example is for treatment of the symptoms associated with amuscular disorder or cardiomyopathy, one may select from among a numberof transgenes associated with muscular dystrophies and/orcardiomyopathies. Examples of suitable genes include, a sarcoglycanprotein (e.g., α, β, or δ, or γ), a Muscular Dystrophy protein(dystrophin or utrophin), a minidystrophin or microdystrophin protein[See, e.g., Y. Yue, et al, Circulation, 108:1623 (September 2003),e-publ. Sep. 2, 2003], calpain, a congenital/limb Girdle MuscularDystrophy protein (Fukutin, Fukutin-related protein, telethonin, orlaminin). Other suitable genes may include beta adrenergic receptorkinase 1 (bARK1) and inhibitors of binding between cardiac myocyteadrenergic receptors and a protein of the Gq subclass. Still other genesinclude, e.g., carnitine palmityl transferase (CPT) 1 and CTP2, which isimplicated in CPT deficiency; dysferlin, which is implicated inlimb-girdle MD type 2B and Miyoshi myopathy; thymidine phosphorylase;SMN2 (SMNC), which is implicated in spinal muscular atrophy; andinsulin-like growth factor (e.g., Igf1), among others. Still other genesinclude SERCA and phoshpholambin, which are implicated incardiomyopathies.

In another embodiment, a transgene may be selected from among transgenesfor which expression from a target cell is desired. Such productsinclude those used for treatment of hemophilia, including hemophilia B(including Factor IX) and hemophilia A (including Factor VIII and itsvariants, such as the light chain and heavy chain of the heterodimer andthe B-deleted domain; U.S. Pat. No. 6,200,560 and U.S. Pat. No.6,221,349).

For example, the Factor VIII gene codes for 2351 amino acids and theprotein has six domains, designated from the amino to the terminalcarboxy terminus as A1-A2-B-A3-C1-C2 [Wood et al, Nature, 312:330(1984); Vehar et al., Nature 312:337 (1984); and Toole et al, Nature,342:337 (1984)]. Human Factor VIII is processed within the cell to yielda heterodimer primarily comprising a heavy chain containing the A1, A2and B domains and a light chain containing the A3, C1 and C2 domains.Both the single chain polypeptide and the heterodimer circulate in theplasma as inactive precursors, until activated by thrombin cleavagebetween the A2 and B domains, which releases the B domain and results ina heavy chain consisting of the A1 and A2 domains. The B domain isdeleted in the activated procoagulant form of the protein. Additionally,in the native protein, two polypeptide chains (“a” and “b”), flankingthe B domain, are bound to a divalent calcium cation.

In some embodiments, the minigene comprises first 57 base pairs of theFactor VIII heavy chain that encodes the 10 amino acid signal sequence,as well as the human growth hormone (hGH) polyadenylation sequence. Inalternative embodiments, the minigene further comprises the A1 and A2domains, as well as 5 amino acids from the N-terminus of the B domain,and/or 85 amino acids of the C-terminus of the B domain, as well as theA3, C1 and C2 domains. In yet other embodiments, the nucleic acidsencoding Factor VIII heavy chain and light chain are provided in asingle minigene separated by 42 nucleic acids coding for 14 amino acidsof the B domain [U.S. Pat. No. 6,200,560].

As used herein, a therapeutically effective amount is an amount ofmacromolecular complex (e.g. a rAAV) that produces sufficient amounts ofFactor VIII to decrease the time it takes for a subject's blood to clot.Generally, severe hemophiliacs having less than 1% of normal levels ofFactor VIII have a whole blood clotting time of greater than 60 minutesas compared to approximately 10 minutes for non-hemophiliacs.

The present invention is not limited to any specific Factor VIIIsequence. Many natural and recombinant forms of Factor VIII have beenisolated and generated. Examples of naturally occurring and recombinantforms of Factor VIII can be found in the patent and scientificliterature including, U.S. Pat. No. 5,563,045, U.S. Pat. No. 5,451,521,U.S. Pat. No. 5,422,260, U.S. Pat. No. 5,004,803, U.S. Pat. No.4,757,006, U.S. Pat. No. 5,661,008, U.S. Pat. No. 5,789,203, U.S. Pat.No. 5,681,746, U.S. Pat. No. 5,595,886, U.S. Pat. No. 5,045,455, U.S.Pat. No. 5,668,108, U.S. Pat. No. 5,633,150, U.S. Pat. No. 5,693,499,U.S. Pat. No. 5,587,310, U.S. Pat. No. 5,171,844, U.S. Pat. No.5,149,637, U.S. Pat. No. 5,112,950, U.S. Pat. No. 4,886,876, WO94/11503, WO 87/07144, WO 92/16557, WO 91/09122, WO 97/03195, WO96/21035, WO 91/07490, EP 0 672 138, EP 0 270 618, EP 0 182 448, EP 0162 067, EP 0 786 474, EP 0 533 862, EP 0 506 757, EP 0 874 057, EP 0795 021, EP 0 670 332, EP 0 500 734, EP 0 232 112, EP 0 160 457, Sanberget al., XXth Int. Congress of the World Fed. Of Hemophilia (1992), andLind et al., Eur. J. Biochem., 232:19 (1995).

Nucleic acids sequences coding for the above-described Factor VIII canbe obtained using recombinant methods or by deriving the sequence from avector known to include the same. Furthermore, the desired sequence canbe isolated directly from cells and tissues containing the same, usingstandard techniques, such as phenol extraction and PCR of cDNA orgenomic DNA [See, e.g., Sambrook et al]. Nucleotide sequences can alsobe produced synthetically, rather than cloned. The complete sequence canbe assembled from overlapping oligonucleotides prepared by standardmethods and assembled into a complete coding sequence [See, e.g., Edge,Nature 292:757 (1981); Nambari et al, Science, 223:1299 (1984); and Jayet al, J. Biol. Chem. 259:6311 (1984).

Furthermore, the invention is not limited to human Factor VIII. Indeed,it is intended that the present invention encompass Factor VIII fromanimals other than humans, including but not limited to, companionanimals (e.g., canine, felines, and equines), livestock (e.g., bovines,caprines and ovines), laboratory animals, marine mammals, large cats,etc.

The complexes may contain a nucleic acid coding for fragments of FactorVIII that is itself not biologically active, yet when administered intothe subject improves or restores the blood clotting time. For example,as discussed above, the Factor VIII protein comprises two polypeptidechains: a heavy chain and a light chain separated by a B-domain that iscleaved during processing. As demonstrated by the present invention,co-tranducing recipient cells with the Factor VIII heavy and lightchains leads to the expression of biologically active Factor VIII.Because, however, most hemophiliacs contain a mutation or deletion inonly one of the chain (e.g., heavy or light chain), it may be possibleto administer only the chain defective in the patient to supply theother chain.

Examples of other transgene products include myostatin inhibitors,insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH),growth hormone releasing factor (GRF), follicle stimulating hormone(FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG),vascular endothelial growth factor (VEGF), angiopoietins, angiostatin,granulocyte colony stimulating factor (GCSF), erythropoietin (EPO),connective tissue growth factor (CTGF), basic fibroblast growth factor(bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor(EGF), platelet-derived growth factor (PDGF), insulin growth factors Iand II (IGF-I and IGF-II), any one of the transforming growth factor αsuperfamily, including TGFα, activins, inhibins, or any of the bonemorphogenic proteins (BMP) BMPs 1-15, any one of theheregluin/neureguin/ARIA/neu differentiation factor (NDF) family ofgrowth factors, nerve growth factor (NGF), brain-derived neurotrophicfactor (BDNF), neurotrophins NT-3 and NT-4/5, ciliary neurotrophicfactor (CNTF), glial cell line derived neurotrophic factor (GDNF),neurturin, agrin, any one of the family of semaphorins/collapsins,netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrins, noggin,sonic hedgehog and tyrosine hydroxylase.

Other suitable proteins as for delivery by the method of the inventionwill be readily apparent. Similarly, transgenes encoding proteins thatare expressed on the cell surface of the targeted cells can be deliveredby the method of the invention.

C. Regulatory Sequences and Construction of Macromolecular Complexes

Suitably, macromolecular complexes carrying transgenes further containregulatory sequences operably linked to the encoded gene product. Inaddition to the major elements identified above, the macromolecularcomplex (e.g., a vector) also includes conventional control elementsthat are operably linked to the transgene in a manner that permits itstranscription, translation and/or expression in a cell transfected withthe macromolecular complex.

As used herein, “operably linked” sequences include both expressioncontrol sequences that are contiguous with the gene of interest andexpression control sequences that act in trans or at a distance tocontrol the gene of interest.

Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation (polyA) signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhancesecretion of the encoded product. A great number of expression controlsequences, including promoters that are native, constitutive, inducibleand/or tissue-specific, are known in the art and may be utilized.

In one embodiment, the regulatory sequences are optimized for expressionin the muscle and/or comprise tissue-specific promoters. For instance,if expression in skeletal muscle is desired, a promoter active in musclecan be used. These include the promoters from genes encoding skeletalβ-actin, myosin light chain 2A, dystrophin, muscle creatine kinase, aswell as synthetic muscle promoters with activities higher thannaturally-occurring promoters (see Li et al., Nat. Biotech., 17:241-245(1999)). However, one of skill in the art can readily select a suitableconstitutive, inducible, or regulated promoter.

Examples of constitutive promoters include, without limitation, theretroviral Rous sarcoma virus (RSV) LTR promoter (optionally with theRSV enhancer), the cytomegalovirus (CMV) promoter (optionally with theCMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], theSV40 promoter, the dihydrofolate reductase promoter, the β-actinpromoter, the phosphoglycerol kinase (PGK) promoter, and the EF1promoter [Invitrogen]. Inducible promoters allow regulation of geneexpression and can be regulated by exogenously supplied compounds,environmental factors such as temperature, or the presence of a specificphysiological state, e.g., acute phase, a particular differentiationstate of the cell, or in replicating cells only. Inducible promoters andinducible systems are available from a variety of commercial sources,including, without limitation, Invitrogen, Clontech and Ariad. Manyother systems have been described and can be readily selected by one ofskill in the art. Examples of inducible promoters regulated byexogenously supplied compounds, include, the zinc-inducible sheepmetallothionine (MT) promoter, the dexamethasone (Dex)-inducible mousemammary tumor virus (MMTV) promoter, the T7 polymerase promoter system[WO 98/10088]; the ecdysone insect promoter [No et al, Proc. Natl. Acad.Sci. USA, 93:3346-3351 (1996)], the tetracycline-repressible system[Gossen et al, Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)], thetetracycline-inducible system [Gossen et al, Science, 268:1766-1769(1995), see also Harvey et al, Curr. Opin. Chem. Biol., 2:512-518(1998)], the RU486-inducible system [Wang et al, Nat. Biotech.,15:239-243 (1997) and Wang et al, Gene Ther., 4:432-441 (1997)] and therapamycin-inducible system [Magari et al, J. Clin. Invest.,100:2865-2872 (1997)]. Other types of inducible promoters that may beuseful in this context are those that are regulated by a specificphysiological state, e.g., temperature, acute phase, a particulardifferentiation state of the cell, or in replicating cells only.

In another embodiment, the native promoter for the transgene will beused. The native promoter may be preferred when it is desired thatexpression of the transgene should mimic the native expression. Thenative promoter may be used when expression of the transgene must beregulated temporally or developmentally, or in a tissue-specific manner,or in response to specific transcriptional stimuli. In a furtherembodiment, other native expression control elements, such as enhancerelements, polyadenylation sites or Kozak consensus sequences may also beused to mimic the native expression.

Methods for assembling and producing a variety of differentmacromolecular complexes as defined herein are known to those of skillin the art and have been described in textbooks and in the literature.See, e.g., Sambrook et al, Molecular cloning: A Laboratory Manual, ColdSpring Harbor Press, Cold Spring Harbor, N.Y. (1989). Selection andproduction of the macromolecular complex is not a limitation of thepresent invention.

II. Transferring a Macromolecular Complex to a Limb of a Subject.

In one embodiment, the method of the invention is used to deliver amacromolecular complex to an arm or a leg of a subject. In order toeffectively deliver the complex according to the invention, thevasculature of the limb is isolated.

The limb is exsanguinated and sufficient pressure is applied to isolatethe limb. In one embodiment, a limb is isolated by applying pressure ata girdle between the limb and the trunk of the subject's body.Typically, this is accomplished by inflating a tourniquet at the pelvicring in order to isolate a leg and/or inflating a tourniquet at theshoulder girdle in order to isolate an arm and fluid is removed throughuse of an elastic band (e.g., an Esmark wrap) that is applied in spiralfashion starting at the foot or hand and wrapping proximally underforce. In another embodiment, two or more of the subject's limbs can beisolated simultaneously. For example, pressure can be applied in orderto simultaneously isolate both legs from the upper portion of the body.Typically, this is accomplished by use of a tourniquet that is placedaround the trunk infrarenally. In still another embodiment, an arm and aleg are isolated at the same time, using the techniques describedherein. Methods of applying pressure to isolate the circulation of alimb or limbs are known to those of skill in the art and are not alimitation on the present invention.

Once the desired limb and/or limbs are isolated and exsanguinated, themacromolecular complex is infused into each limb at a high hydrostaticpressure. It is anticipated that efficiency of gene transfer to a limbwill increase as the pressure increases through the range providedherein. For example, in a small mammal, efficiency of gene transfer hasbeen demonstrated to increase as the pressure increases through therange 100, 200, and 400 torr.

Exsanguination and infusion can be readily accomplished using asuitable, commercially available, balloon catheter located in a suitablevessel of the limb (e.g., a vein or artery). In one embodiment, it isdesirable to utilize a balloon tip catheter placed percutaneously usinga dilator to maximize the bore available. Optionally, it may bedesirable to have a second inflatable tourniquet placed just proximal tothe catheter insertion site, distal to inflated balloon tip to minimizeleak around catheter at wall of vein. In another embodiment, it isdesirable to utilize a larger bore non-balloon tip catheter placedthrough a very small cut down (1.5 cm incision).

Typically, the macromolecular complex is infused in a physiologicallycompatible solution. In one embodiment, the solution containsphysiologic solution that may be readily selected from among saline,isotonic dextrose, or a glycerol solution, among others that will beapparent to one of skill in the art given the information providedherein. In one embodiment, the physiologic solution is oxygenated inorder to maximize the safety margin and minimize the risk of limbischemia. However, the invention is not so limited. Suitably, the totalvolume of the solution infused into the limb is in the range of 20 to100%, 25 to 90%, 30 to 80%, 40 to 70%, 50 to 60%, of the estimatedvolume of the extremity. The concentration of the macromolecular complexin the solution can vary depending upon the type of complex selected.However, given the information provided in the present invention, one ofskill in the art can readily select higher or lower volumes.

The limb is resanguinated in a conventional manner, e.g., by loweringthe pressure in the proximal tourniquet.

III. A Method of Transferring a Macromolecular Complex Systemically

In one embodiment, the present invention permits the transfer of amacromolecular complex as defined herein to extravascular tissuesystemically. In one particularly desirable embodiment, systemicdelivery involves separating the central circulation, i.e., all vesselsdirectly supplying the thoracic and abdominal viscera, from theperipheral circulation, i.e., vessels supplying the skeletal muscles.The method involves restricting the flow of fluids through the centralcirculation using the balloon catheter of the invention. Thus, themethod is particularly well suited for targeted delivery of aheterologous molecule to muscle cells of a subject, while avoidingdelivery to the organs. This is particularly advantageous where deliveryto a selected organ, e.g., the liver or lung, is undesirable in view ofthe selected transgene, the selected vector, or some other component ofa heterologous molecule.

According to the present invention, a patient is placed under totalcirculatory arrest. Typically, this is performed as follows. The patientis placed under general endotracheal anesthesia, heparin is administeredand total circulatory arrest is achieved by way of a carotid-jugular orfemoral cannulation using a pump oxygenator. Known techniques are usedto accomplish the placement of these conventional cannulae in suitablevessels, e.g., in the carotid artery and/or in a femoral artery.Conventional heart-lung cannulae are utilized.

For example, one catheter may be threaded from a femoral position (e.g.,a femoral artery) and through the aorta. Alternatively, the catheter maybe threaded from the carotid artery in a superior-to-inferior directionthrough the aorta. A second catheter may be threaded into the vena cavain either a superior-to-inferior director or in an inferior-to-superiorto direction. When the catheter is inserted in the superior-to-inferiordirection, it is preferably inserted into a human subject through theright jugular vein or through another vein which communicates with thesuperior vena cava. When the catheter is inserted in theinferior-to-superior direction, it is preferably inserted into a humansubject through a femoral vein of the subject, another it may beinserted through a vein that communicates with the inferior vena cava.Alternatively, the catheter may be threaded entirely through the subject(e.g., extending from both the subject's femoral vein and the subject'sjugular vein).

The patient is rendered hypothermic, in the range of 15 to 18° C. usingthe protocols described previously for pediatric cardiac surgery andadult aortic arch reconstruction. See, e.g., U.S. Pat. No. 6,492,103,“System for organ and tissue preservation and hypothermic bloodsubstitution”. The patient is then partially exsanguinated anddecannulated from the first and second heart-lung cannulae.

One balloon catheter of the invention is inserted into one of thecannulation sites and threaded so that upon inflation, the balloonoccludes both the superior vena cava and inferior venae cava. Anotherballoon catheter of the invention is inserted into the other cannulationsite and threaded through the aorta to a position just adjacent to theaortic arch. Desirably, this internal occlusion balloon runs from theaortic bifurcation to the aortic arch. Proper positioning of thesecatheters may involve fluoroscopy or ultrasound techniques such as areknown to those of skill in the art. Once positioned properly, theballoon catheters are inflated so that one balloon catheter occludes thevenae cavae and the second balloon catheter occludes the aortic space.

Typically, the catheters are inflated to a pressure exceeding thatapplied from cannulae in the extremities of the patient. In oneembodiment, the macromolecular complex is delivered in a physiologicsolution as described above. In one embodiment, the solution isoxygenated. In one embodiment, the solution is an oxygenated,physiologic saline. Suitably, the complex in solution is then infused toall four of the patient's extremities via the cannulae located therein.

The solution can be infused under high pressure, as defined herein.Alternatively, the pressure in the microvascular space can be reducedbelow physiological levels by the catheters and the exsanguinationprocedure. Thereafter, as pressure/volume are increased toward baselinepressure/volume via delivery from the cannulae at the extremities,delivery to extravascular tissue will increase. Typically, about 0.5 to4 liters of liquid are delivered according to this method. However,smaller or larger amounts can be readily used. For example, in a 70 kgadult, about 1.5 to 2 liters are suitable for an isolated limb and 3 to4 liters should be well tolerated systemically. In a small child, about0.5 liters to 1 liter can be used in an isolated limb and 1 to 2 literscan be used systemically. However, these amounts can be readily adjustedby one of skill in the art, taking into consideration the informationprovided herein and that which is known to one of skill in the art.

Following dissipation of the initial pressure gradient, typically about2 minutes, in one embodiment, the solution is allowed to dwell.Typically, the fluid is permitted to dwell for about 30 seconds to 30minutes, taking into consideration such factors as whether this dwellperiod is being utilized during system delivery or delivery to a limb asdescribed above. For example, a suitable dwell period for systemdelivery may be from about 30 second to 1 minute, to about 90 seconds,or longer. However, longer dwell periods may be suitable for delivery tothe limb, e.g., from about 1 minute to about 20 minutes, or longer.However, longer or shorter dwell periods may be readily selected giventhe information provided herein.

Optionally, the solution is flushed prior to withdrawing the ballooncatheters and reinsertion of the heart-lung cannulae. The heart-lungcannulae are then reinserted and the patient is then resanguinated andrewarmed until hemodynamically stable. The cannulae are removed and thevessels repaired under direct vision. After closure of the smallincision, the patient is extubated after weaning parameters are met. Inthe post procedural period, there is an anticipated need for oxygensupport and hemodynamic monitoring as residual fluid in the interstitiumis progressively mobilized and excreted by way of the kidneys. For largevolume loads, hemofiltration may be required. Optionally, hemofiltrationcan be instituted in the operating room before decannulation. The methodof the invention may involve subjecting the animal to extracorporealcirculatory support and oxygenation. Preferably, a heart-lung machine isused according to methods known in the art. Extracorporeal circulatorysupport and oxygenation permits blood flow to the lungs of the animal tobe minimized, thus minimizing exudation from the pulmonary blood vesselsof the animal into the lungs.

A method of subjecting a human to extracorporeal circulatory support andoxygenation has been described in U.S. Pat. No. 6,177,403. For example,an extracorporeal lung support (ECLS) pump oxygenator can be connectedto a pair of cannulae inserted into the human, where one cannula extendsinto the right atrium of the human, and the other cannula extends intothe aorta of the human. Blood is withdrawn from the right atrium,oxygenated extracorporeally, and returned to the atrium of the human ata controlled pressure and flow rate. Using this method, blood flow tothe lungs is minimized, and exudation from pulmonary blood vessels intothe parenchyma of the lungs is minimized. Hepatic blood flow in thehuman may also be occluded.

Because an oxygen-transporting agent is provided to the vessel, thevessel can remain occluded, and the vector and agents can remain withinthe vessel for an extended period. In those embodiments in which aclearance solution is provided to the vessel, excess vector and agentsare removed from the vessel prior to re-establishing systemic bloodcirculation in the animal, thereby minimizing any potential undesirableeffects caused by the presence of the vector or agents in an area of theanimal's body other than the vessel.

Whole blood, temporarily retained the venous reservoir of a modified(enlarged venous reservoir) pump-oxygenator, is reinfused to the patientvia the oxygenator and the arterial cannula, while saline is removedfrom the venous cannula and cycled through a red blood cell recoverydevice (such as the commercially available “Cell Saver” device). Thisprocess is continued until the patient's entire blood volume is restoredfrom the venous reservoir. After this point, the entire erythrocyte massof the recovered saline perfusate is spun down and reinfused asappropriate, during a process of ongoing hemoconcentration byhemofiltration. Once the hematocrit is estimated to be aboveapproximately 15%, warming begins by way of the oxygenator and continuesuntil the patient reaches 37° C.

Although the method of the invention is particularly well suited fordelivery of heterologous macromolecular complexes to target cellswithout utilizing vascular permeability-enhancing agents, one of skillin the art may utilize the devices and methods of the present inventionin combination with such agents. Such agents include, e.g., histamine,acetylcholine, an adenosine nucleotide, arachidonic acid, bradykinin,cyanide, endothelin, endotoxin, interleukin-2, ionophore A23187,nitroprusside, a leukotriene, an oxygen radical, phospholipade, plateletactivating factor, protamine, serotonin, tumor necrosis factor, vascularendothelial growth factor, a venom, and a vasoactive amine. See, e.g.,WO 99/31982, Jul. 1, 1999. Alternatively, other methods for targeteddelivery to the heart may be utilized.

In addition, the methods of the invention may be used in combinationwith conventional delivery of other active ingredients or other methods.For example, it may be desirable to perform the method of the inventionin a regimen that involves sequential delivery of a desired heterologousmolecule to the cardiac muscle by targeted delivery to the heart. See,e.g., WO 99/59666.

In another embodiment, the invention provides a method described fortargeted delivery to the heart muscle. Notably, in an experimentalseries, the inventors showed that retrograde perfusions of the heart viathe coronary veins, followed by heterotopic transplantation can besuccessfully utilized to eliminate the need for the use of aninflammatory mediator or vasodilator to achieve highly efficient genetransfer. Implementation of this in the heart in situ requiresseparation of the coronary and systemic circulations, using in apreferred embodiment a modification of the system previously detailed[Bridges, et al, Annals of Thoracic Surgery, 73:1939-1946 (2002),incorporated by reference] coupled to retrograde infusion. In thepresent context, the coronary and systemic circulation are as describedin Bridges, et al et al. and this aspect of the invention involvesisolating the cardiac circulation from the remainder of the patient'scirculatory system. In this embodiment, the heart is cooled to about 15to 18° C., but the patient is not required to undergo completeexsanguination. Nor is the remainder of the patient subjected tohypothermic conditions. Suitably, the systemic circulation of thepatient is provided with constant, high level, oxygenated fluid usingtechniques known for use in cardiopulmonary procedures.

See, FIG. 7, which illustrates the important components of the cardiacisolation method of the invention, including the aortic cross-clamp 11,superior vena cava (SVC) and inferior vena cava (IVC) cannulae 13 and15, respectively, which are connected to a systemic pump oxygenator 17,which returns blood to the patient's femoral and/or carotid arteriesthrough a cannula 19. The components also include SVC 21 and IVC 23snares and a pulmonary cross-clamp 25. Retrograde perfusion takes placein a recirculating pathway 27, through a coronary sinus catheter 29 andthrough a cardiac arterial in-flow catheter 21, a right ventricular (RV)catheter 33 and a left ventricular (LV) vent catheter 35, which havebeen described in Bridges, et al. The present invention further utilizesa coronary sinus catheter that is inserted into the right atrium andinto the coronary sinus to achieve retrograde perfusion. The leftpulmonary veins 37 and 29 and right pulmonary veins 41 and 43 aredepicted for perspective. Using these techniques, the cardiaccirculation is infused with a heterologous molecule such as has beendescribed herein and the use of retrograde perfusion permits high levelsof transfer into the venous interstitium, thereby enhancing transferinto the cardiac muscle as compared to methods known in the art andavoiding transfer of the heterologous molecule to the remainder of thepatient.

IV. Balloon Catheter

In one embodiment of an internal occlusion balloon catheter inaccordance with the invention, the balloon extends from a point adjacentthe distal end of the cannula (i.e., the end which first enters thepatient through a cannulation site) to a point adjacent the proximal endof the cannula. This balloon catheter differs from conventional ballooncatheters, in which the balloon is typically situated at the distal endof the cannula, and also from other balloon catheters, in which theballoon is situated at or near the proximal end or in which balloons aresituated at both the distal and proximal ends, with a significantseparation between them. The balloon in accordance with the inventionmay take the form of a single, elongated, tubular envelope having acontinuous internal space for expansion fluid, or alternatively, anelongated envelope having plural internal compartments or segments,isolated from one another by radial membranes which connect the outerpart of the balloon envelope to the cannula, thereby anchoring theballoon envelope against axial translation along the length of thecannula. In each case, the balloon envelope, when inflated, that is,when expanded by a suitable expansion fluid, is an elongated,substantially continuous, cylindrical tube having rounded ends, andextends from a location adjacent the distal end of the cannula, towardthe proximal end throughout an appropriate distance such that theballoon, when inflated can substantially fill the blood vessel intowhich it is inserted throughout the entire length of the vessel.

When the balloons of the catheters are inflated within the aorta or venacavae, they initially encounter relatively little resistance as thewalls of the vessels expand. However, when the vessel reaches theirfully expanded condition, the collagen component resists furtherexpansion, and the vessel walls then exert a counter pressure on theballoons. In general, the balloons should be made of a material having avery high degree of distensibility so that they can exert pressure onall parts of the walls of the vessels in which they are situated,expanding the vessels to the point at which further expansion isresisted by the collagen in the vessel walls, and thereby fullyoccluding the vessels. However, as explained below, in the case of aballoon catheter for insertion into the vena cavae, the portion of theballoon that is ultimately situated in the right atrium of the heart issufficiently limited in its distensibility to avoid overdistension of,and resultant damage to, the heart. In one embodiment, a ventingcatheter is placed in the right atrium to relieve pressure.

In this embodiment, the balloon catheters extend substantially the fulllength of the vessels in which they are situated in order to facilitatecompartmentalization of the circulation in the central and peripheralvascular systems. The central vascular system comprises named vesselsdirectly supplying the thoracic and abdominal viscera, and theperipheral vascular system comprises named vessels supplying theskeletal muscle mass. When, in the process of delivering themacromolecular complex, high venous pressure is applied to theperipheral circulation, the pressure within the balloon catheterstransiently restricts flow from the peripheral to the centralcirculation. Transient separation of the central and peripheralcirculation promotes efficient vector delivery to the skeletal muscleinterstitium, while the heart is protected from overdistension. Inaddition, vector transport to the abdominal viscera is minimized by therestriction of flow through the aorta and vena cavae, as theyinterconnect the named vessels supplying the thoracic and abdominalviscera.

Those skilled in the art can select from among various known materials,shapes and designs for the catheter. The catheter comprises an elongatedshaft which will ordinarily be hollow so that it serves as a cannula.The cannula is preferably formed of a flexible thermoplastic material, athermoplastic elastomer or a thermoset elastomer. The cannula may befabricated from components separately extruded and joined togetherend-to-end, for example by heat welding or by adhesive bonding. Thecannula may also be fabricated by dipping, or by composite constructiontechniques, in which separately fabricated components are joinedtogether. Alternatively, the entire cannula may be fabricated as a unit.Suitable materials for the elongated catheter shaft include, but are notlimited to, polyvinylchloride (PVC) polyurethane, polyethylene,polypropylene, polyamides (nylons), polyesters, silicone, latex, andalloys or copolymers thereof, as well as braided, coiled or counterwoundwire or filament-reinforced composites.

Of course, many other general catheter designs are known to those ofskill in the art, and can find useful application within the context ofthe invention. See, e.g., WO 99/59666, Nov. 25, 1999.

FIG. 1 depicts an embodiment of the balloon catheter 101, designed forinsertion into the aorta. In order to facilitate placement of thecatheter and to improve the stability of the catheter and therebymaintain it in the proper position in the patient's aorta, a distalregion 102 of the cannula is preshaped in a curve to match the internalcurvature of the patient's aortic arch. The catheter is J-shaped, withits distal region formed in an arcuate curve subtending an angle ofapproximately 180 degrees. In the case of an adult human patient, thearcuate curve should have a radius of curvature of approximately 2 to 4cm, to match the typical curvature of the adult aortic arch. The distalend of the cannula may be skewed slightly out of the plane of the curveto accommodate the angle of the patient's ascending aorta. Additionally,the cannula (catheter shaft) may be reinforced, particularly in thecurved distal region, for example by braided or coiled wire, to improvethe stability of the catheter still further, and thereby ensure that itcan be maintained in the proper position in the patient's aorta.

As shown in FIG. 1, an inflatable balloon 103 surrounds the cannula, andis secured to the cannula at a point adjacent the distal end of thecannula (i.e., the end which first enters the patient through thecannulation site), and also at a point spaced by a suitable distancefrom the distal end. In some cases, the balloon extends to a locationadjacent the proximal end of the cannula. However, in other cases, asdepicted in FIG. 1, a portion of the cannula will extend proximallyrelative to the proximal end of the balloon so that an externallyextending part of the catheter is provided for manipulation. Also, insome applications in accordance with the invention, it may be desirableto have the balloon extend only along a portion of the blood vesselspaced from the catheter entry point. For example, in the case of aballoon catheter intended for occlusion of the aorta, the balloon, maybe located only above the bifurcation where the abdominal aorta branchesinto the iliac arteries, and therefore spaced by a considerable distancefrom the catheter entry point in a femoral artery.

The balloon may be secured to the cannula by any of various knownfastening schemes such as adhesive bonding, heat welding, wrapping witha winding of filamentary material, or combinations of adhesive bondingor heat welding and reinforcing material, or the like. In the case of aballoon having a single, uninterrupted, internal space, the balloon willordinarily be secured only at its distal and proximal ends to thecatheter. However, radial connections in the form of membranes orfilaments may be provided between the cannula and the balloon atintermediate locations. Alternatively, as will be described below, theballoon may comprise a series of separate balloon-like segments disposedalong the shaft in end-to-end relationship to form a balloon that isessentially a single balloon when viewed from its exterior, but which iscomposed of plural compartments isolated from one another by membranesformed by the ends of the balloons where they meet each other. In all ofthe cases in which the balloon is attached to the cannula atintermediate points as well as at the ends of the balloon, the multipleattachment sites assist in avoiding longitudinal movement of theinflated balloon, which may be caused by inflation and/or backflow.

The cannula contains an interior lumen for conducting an expansion fluidto the interior of the balloon. A conventional balloon catheter isprovided with fluid conducting apertures located at its distal end forconducting expansion fluid from the interior lumen to the exterior ofthe cannula for inflation of the balloon. However, because the length ofthe balloon of the invention is much longer than that of conventionalballoons, the cannula may have multiple apertures located along itslength. Optionally, a series of such apertures may be placed inproximity to one another and spaced apart from another series ofapertures. Typically, a series of apertures is composed of two or threeapertures that are spaced apart by 3 to 10 mm. In one embodiment, theapertures are disposed in longitudinally spaced relationship along thelength of the cannula Alternatively, a series of apertures may belocated at a single station along the length of the cannula, beingdisposed around the circumference thereof. Longitudinally spaced groupsof apertures are used in embodiments incorporating a compartmentalizedballoon to provide for simultaneous or sequential inflation of thecompartments, as described below.

The cannula may have multiple lumens. In addition to at least one lumenfor carrying a suitable expansion fluid for inflation of the balloon103, the shaft may also have a corporeal perfusion lumen, an archperfusion lumen, an oxygenation lumen, a guide wire and cardioplegialumen, and a root pressure lumen.

In the embodiment illustrated in FIG. 1, the catheter is provided with acommon balloon inflation lumen which extends through the shaft from theproximal end to proximal balloon inflation apertures 105, and continuesto distal balloon inflation apertures 107, the proximal and distalinflation apertures being disposed within the balloon, respectively nearthe proximal and distal ends of the balloon.

Alternatively, separate balloon inflation lumens may be provided, whichextend through the cannula shaft from the proximal end to differentgroups of balloon inflation apertures. The separate balloon inflationlumens permit greater flexibility and precision in inflation of theballoon, or sequential inflation of compartments or regions in the caseof a segmented balloon.

Optionally, the catheter can be provided with one or more markers, whichmay include radiopaque markers and/or sonoreflective markers, to enhanceimaging of the catheter by fluoroscopy or ultrasound used to monitor theposition and placement of the catheter. Typically, these markers areplaced along the distal end of the catheter. Suitable materials for suchmarkers are well known to those skilled in the art, and include, forexample, a ring of dense radioopaque metal, such as gold, platinum,tantalum, tungsten or alloys thereof, or a ring of a polymer or adhesivematerial heavily loaded with a radiopaque filler material.

In the embodiment illustrated in FIG. 2, the balloon 111 is composed ofa series of balloon segments, including segments 113-123, each segmentbeing a complete balloon in itself. These segments are disposed alongthe cannula 125 in end-to-end relationship, their ends meeting oneanother and forming double-walled radial membranes shown, for example at127 and 129. The membranes isolate the interiors of the balloon segmentsfrom one another, and reinforce the balloon structure, resistinglongitudinal movement of the balloon along the length of the catheter.Groups of apertures in the cannula 125 are disposed to deliver expansionfluid to the interiors of the individual balloon parts. For example,aperture groups 131 and 133 are positioned to deliver expansion fluid toballoons 113 and 115, respectively. Optionally, the groups of aperturesmay be in communication with separate inflation lumens within thecatheter shaft, thereby allowing controlled inflation of the segments ofthe balloon. For example, the segments may be inflated sequentially,segment 113 at the distal end of the balloon being inflated first,followed by inflation of segment 115 and then part 117, and so on, untilinflation of the balloon segment 123 at the proximal end is achieved.Alternatively, it may be desirable to inflate the segments at the distaland proximal ends, following by inflation of the segments in the regionsbetween the two ends. The balloon may, of course be composed of anydesired number of such segments, and the segments may be inflatedsimultaneously through a single inflation lumen, or separately throughdifferent inflation lumens.

In the collapsed state, the balloon fits and conforms to the cannula, sothat the diameter of the cannula/balloon combination, when inserted, isonly greater than that of the cannula by twice the relaxed balloon wallthickness. In its inflated state, the balloon expands to a diametersufficient to occlude blood flow in the vessel into which it isinserted. Preferably, the balloon has an inflated length that does notchange significantly as compared to its noninflated length. This isespecially significant in the case of the aortic arch, wherelongitudinal expansion could cause damage.

In the case of a balloon catheter intended for use in the aorta, theballoon may extend substantially the entire length of the aorta, asshown in FIG. 3, including the aortic arch. Thus, as shown in FIG. 3,where the catheter shaft 135 is inserted through a femoral artery, theballoon 138 extends from a location adjacent the bottom of the abdominalaorta, through the aortic arch, and into the ascending aorta, therebysubstantially filling the entire aorta. As will be apparent from FIG. 3,the occlusion of the entire aorta, effectively prevents cross flow,through the aorta, between the various branch vessels, including thebranches leading from the aortic arch, the intercostal arteries, and thelumbar and celiac arteries as well as the superior and inferiormesenteric arteries.

In the case of the vena cavae, as shown in FIG. 4, the balloon catheterpreferably comprises a cannula 140, with a balloon consisting of plural,separately inflatable segments in end-to-end relationship, in anarrangement similar to that shown in FIG. 2. In the case of FIG. 4, theballoon comprises three balloon segments 142, 144, and 146. The catheteris preferably inserted through the right jugular vein, and extendsthrough the superior vena cava, and through the right atrium of theheart, to the lower end of the inferior vena cava.

The proximal balloon segment 142 and the distal balloon segment 146 arecomposed of a highly distensible polymeric material, as in the case ofthe aortic balloon catheters of FIGS. 1-3. The intermediate balloonsegment 144, on the other hand, should have more limited expansibilityin order to prevent overdistension of right atrium. Overdistension cancause unraveling the titin in the wall of the right atrium, resulting ina dysfunction of the heart when restarted at the conclusion of themacromolecular transfer procedure. This dysfunction can seriously impedepatient recovery.

Overdistension can be avoided by various measures, including selectionof a less distensible balloon material for the balloon segment 144,constructing balloon segment 144 with a distension-limiting layer orwrapping, or by inflating balloon segment 144 through a separateinflation lumen, and limiting the amount of expansion fluid introducedinto it.

As shown in FIG. 5, the main portion of the inferior vena cava 148 iscomposed of smooth muscle, but the portion 150 of the inferior vena cavathat extends above the boundary 152 where the vessel enters the rightatrium of the heart is composed of striated muscle.

As shown in FIG. 6, balloon segment or compartments 144 should besufficiently long that, when the balloon catheter is in place in thevena cavae, its lower end extends slightly below the boundary 152, andits upper end extends into the superior vena cava 154, but not to thelocation where the azygous vein 156 enters the superior vena cava.Consequently, the superior vena cava is occluded by balloon segment 142at the location of its junction with the azygous vein, but neither ofthe highly distensible balloon segments 142 and 146 extends into theright atrium. Therefore excessive distension of the right atrium isavoided. Balloon segment 144 prevents segments, or compartments, 142 and146 from expanding longitudinally into the right atrium, and occludesshort portions of the vena cavae immediately adjacent the heart. Theballoon segments 142 and 146 prevent cross flow between the variousbranches of the superior and inferior vena cavae, for example, theazygous vein and the hepatic and renal veins, and also prevent coaxialflow of fluid from the vena cavae into the right atrium.

As shown in FIG. 4, a vent catheter 158, threaded through the internaljugular vein, passes alongside the balloon segment 142 and the upperpart of balloon segment 144, into the right atrium. The vent cathetercontinues, through the tricuspid valve, into the right ventricle. Aseries of apertures is provided along the distal portion of the ventcatheter 158 so that some of the apertures are within the right atrium,and others are in the right ventricle, when the vent catheter is inplace. Thus, the vent catheter assists in preventing cardiac distension.Although the vent catheter is shown in FIG. 4 is a separate catheter,the vent catheter can be incorporated into the balloon catheter.

In the case of a catheter intended for use in the aorta of an adultpatient, the inflated outer diameter of the balloon will generally be inthe range from 1.5 to 5.0 cm and the length of the balloon willgenerally be in the range from 40 to 70 cm, and preferably in the rangefrom 50 to 60 cm.

In the case of a catheter intended for use in the aorta of a pediatricpatient, the inflated outer diameter of the balloon will generally be inthe range from 0.5 to 2 cm, and the length of the balloon will generallybe in the range from 20 to 30 cm.

In the case of a catheter intended for use in the aorta of an infant,the inflated outer diameter of the balloon will generally be in therange from 0.3 to 1 cm, and the length of the balloon will generally bein the range from 10 to 20 cm.

In the case of a catheter intended for use in the vena cavae of an adultpatient, the inflated outer diameter of the balloon will generally be inthe range from 2 to 5 cm, and the length of the balloon will generallybe in the range from 40 to 70 cm.

In the case of a catheter intended for use in the vena cavae of apediatric patient, the inflated outer diameter of the balloon willgenerally be in the range from 1 to 2 cm, and the length of the balloonwill generally be in the range from 20 to 30 cm.

In the case of a catheter intended for use in the inferior vena cava ofan infant, the inflated outer diameter of the balloon will generally bein the range from 0.3 to 1 cm, and the length of the balloon willgenerally be in the range from 10 to 20 cm.

Suitable materials for the balloon include materials that exhibitsubstantially identical expansion properties under the hypothermicconditions described herein (about 15 to 18° C.) as at body temperature(about 37° C.). Examples of suitable materials include flexible polymersand elastomers, which include, but are not limited to,polyvinylchloride, polyurethane, polyethylene, polypropylene, polyamides(nylons), polyesters, latex, silicone, and alloys, copolymers andreinforced composites thereof. If the balloon is secured to the cathetershaft by adhesive bonding, the adhesive may be any material compatiblewith the balloon and catheter shaft materials, and may be composed, forexample, of flexible polymers and elastomers, which include, but are notlimited to, polyvinylchloride, polyurethane, polyethylene,polypropylene, polyamides (nylons), polyesters, latex, silicone, andalloys, copolymers and reinforced composites thereof. In addition, theouter surface of the balloon may include a friction increasing coatingor texture to increase friction with the aortic wall when deployed.

V. Clinical Kit

In one aspect, the invention provides a kit for use by a clinician orother personnel. Typically, such a kit will contain a balloon catheterof the invention and, optionally, instructions for performing a methodas described herein. In another embodiment, the kit will contain amacromolecular complex in a physiologically compatible saline solutionand, optionally, instructions for dilution, and performing a method asdescribed herein.

The kit of the invention may also contain an oxygen-transporting agentand/or at least one disposable element of an extracorporeal circulatorysupport and oxygenation system. For example, at least one disposableelement can be an oxygenator having a hollow body, a liquid inlet influid communication with the interior of the body, a liquid outlet influid communication with the interior of the body, a gas inlet forproviding gas to the interior of a gas chamber, at least onegas-permeable membrane separating the gas chamber from the interior ofthe body, and a gas outlet for permitting gas to exit from the gaschamber, whereby gas exchange is enabled between a fluid in the interiorof the body and a gas in the gas chamber. The oxygenator may beconstructed as described in U.S. Pat. No. 6,177,403, wherein thegas-permeable membrane comprises PTFE tubing extending within at least aportion of the tube, and wherein the gas chamber comprises the interiorof the PTFE tubing.

Thus, the kit of the invention may also comprise an oxygen-transportingagent or at least one disposable element of an extracorporealcirculatory support and/or oxygenation system.

A kit that is useful for performing the method of the invention iscontemplated which comprises, in addition to the macromolecular complexand/or balloon catheter of the invention, at least one disposableelement of an extracorporeal circulatory support and oxygenation system.Preferably, such a kit comprises all of the single-use components neededto perform the method of the invention, including a macromolecularcomplex, a vascular permeability-enhancing agent, a fluid deliveryinstrument such as a syringe or a length of peristaltic pump tubing, anda cannula such as a hollow bore needle adapted to fit a syringe. Such akit may also contain a pharmaceutically acceptable carrier, a secondcannula, an oxygen-transporting agent, a clearance solution which issubstantially free of the macromolecular complex, one or more bloodvessel occluding devices, such as a clamp, hemostat, or tourniquet, adisposable oxygenator, and the like.

EXAMPLES

Selection of appropriate models for these experiments by the inventorsreflected the comparative anatomy and morphology of the vascular tree inrodents, carnivores and primates. The seemingly unique absence of valvesin the peripheral veins of mice prompted the exclusion of this speciesfrom these studies on the grounds that the small size might havefacilitated evolutionary loss of the peripheral venous pump mechanism.An additional consideration that prompted the complementary use of onesmall and one large animal model is the previously documented increasein the thickness of the microvascular basal lamina as a function ofpostural hydrostatic pressure. Catheters of the largest bore possiblefor insertion into the distal saphenous veins of the rat and dog,peripheral to and upstream of the valves consistently observed in thelarger veins of the proximal limb, were designed. To facilitatepressurized infusion without leak at the venipuncture sites, snugligatures were placed around the catheters and the veins were ligateddistally. Studies using fluorescently labeled albumin suggested thatrapid infusion against a proximal tourniquet would force homogenousfluid and solute extravasation throughout the limb, creating a volume oftissue edema several times greater than that of the blood volume in theextremity. To address the size-dependency of the forced extravasationprocess, recombinant vectors derived from adenoviruses andadeno-associated viruses were substituted. The marker gene LacZ was usedto facilitate quantitation and visualization of tissue transduction.

Thus, the following examples are illustrative of the present invention,and describe results for several exemplary macromolecular complexes,including two types of viral vectors and a large, charged proteinaceousmolecule (albumin).

The invention is not limited to the methods or apparatus described inthese examples.

Example 1 Evans Blue Dye (EBD) Study

EBD solution was prepared as described in C. R. Bridges, et al, AnnThorac Surg, 73(6):1939-1946 (June 2002). A male adult Fisher ratunderwent hindlimb isolation as described below.

Approximately 0.05 mg/g body weight of EBD was delivered in a sterilePBS & albumin solution. O.16 ml of the solution was diluted to a volumeof 5 ml and infused through the greater saphenous vein with a 400 torrapplied pressure into the isolated hindlimb. 2.4 ml of the solution wasinfused slowly into the contralateral hindlimb without isolation. Therat was necropsied 30 minutes after the start of infusion.

Example 2 Materials

The following materials were used in the studies described in Examples 3and 4, which demonstrate the method in a rat and canine model,respectively.

A. rAAV and rAd

All vector was procured through University of Pennsylvania Vector Coreand was tested to be replication defective and endotoxin negative.

All vector used encoded the marker gene lac-Z coupled to a CMV promoter.Varying lots of vector were used for experiments with the goal ofdelivering ˜10¹³ genome copies (GC)/kg of rAAV and 10¹² particles/kg ofrAd5 in isolated limb infusion studies. The isolated hemibody trialsdelivered ˜5×10¹³ GC/kg of rAAV. rAAV serotype 2/1 was utilized for allexperiments except for preliminary studies in the rat using rAAVserotype 2/2. The rAAV 2/1 used in the dogs was an aggregate pool of 5lots.

B. Tissue Analysis

Tissue samples were ‘snap frozen’ in isopentane cooled in liquidnitrogen at the time of necropsy. 8 micron sections were generated usinga cryotome and then incubated overnight at 37° C. in PBS supplementedwith 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal; Sigma),magnesium chloride, and Fe(II,III)CN (potassium ferro/ferricyanide).After completion of reaction incubation, the tissues werecounter-stained with hematoxylin and eosin, no bluing agent was used.Tissue specimens from rats were whole-mount-stained for lacZ activityafter fixation with 0.2% glutaraldehyde and 2% paraformaldehyde in PBSat necropsy. Protein extracts from the tissues of treated animals wereassayed for β-gal activity using a luminometric kit (Applied Biosystems,Foster City, Calif.) and measured for protein concentration with theBradford reagent (Pierce).

Example 3 Gene Transfer in Rat Model

A. Intravascular Injection of rAAV in Rat Isolated Limbs

After anesthesia with xylene and ketamine, inbred (male adult Fisher344) rats underwent femoral artery and vein isolation with transmuscularplacement of two overlapping 2-0 prolene tourniquets at the level of theproximal thigh. Through a small incision, the greater saphenous vein wascannulated with heat-tapered polyethylene tubes (PE 10; BectonDickinson, Sparks, Md.) attached at their other ends to 30-gauge needleswas performed. After tourniquets were tightened and the limb circulationwas isolated, a total volume of 5 ml of sterile PBS and 10¹² GC of rAAVencoding the lac-z marker gene was infused at pressures of 100, 200, 400torr. The infusion time varied with delivery pressure, but the totaltime the rAAV solution was left in place was 30 minutes from thebeginning of infusion. The tourniquets were then released and thecatheter removed. The saphenous vein was ligated with a silk suture andthe incision was closed with resorbable suture. The same procedure wasfollowed without tourniquet placement to approximate zero torr pressureinfusions. Realizing the volume and rate of infusion may have an impacton intravascular pressure, we performed slow continuous infusions of 5ml and 0.5 ml with the same quantity of rAAV, 10¹² GC. A simpleintramuscular (i.m.) injection of rAAV was delivered to the tibialisanterior muscle on the contralateral limb as a positive control.

The results with at least two animals in each group showed that both AAV2 and AAV 1 vectors efficiently transduced muscle fibers throughout thelimb and that the higher the pressure used the greater the efficiency oftransduction.

B. Assessment of Adenovirus (Ad Vector) and Intravascular Injection ofrAAV in Rat Lower Hemibody

The protocol described in Part A was subsequently modified in threeways: 1) substitution of recombinant adenovirus vector for AAV, 2)simultaneous infusion of AAV vector at 200 torr into one hindlimb andone forelimb with proximal tourniquets at both limb girdles, and 3)simultaneous infusion of AAV vector at 200 torr into both hindlimbsagainst an extracorporeal tourniquet placed at the mid-abdomen. For 3),adult Fisher 344 male rats underwent lower hemi-body delivery of thetransgene after cannulation of both hindlimb saphenous veins. Twice thegenomic copies of the vector was diluted in 10 ml of sterile PBS anddelivered at 200 torr after atraumatic tourniquet placement at the levelof the infraumbilical abdomen. Necropsy was performed at day 14.

All three of these modifications were well tolerated by the rats, asjudged by their rapid and complete recovery of normal patterns ofactivity including normal voluntary movement of all four extremities.Findings at completion of the experiments (7 days for #1, 4 weeks for #2and 3), were notable for high-level marker gene expression indicative ofefficient vector transduction among the majority of muscle fibersthroughout the muscles below the level of the tourniquets in eachexperiment.

Both the pattern of tissue transduction and the total amount oftransgene product detected depended entirely on the method of infusionused. Simple low pressure infusion of vector into a peripheral veinresulted in control (i.e., indistinguishable from that in uninjectedanimals) levels of muscle transduction throughout the body. In contrast,moderate (100 torr) and high pressure (400 torr) infusion of the sametotal dose of vector, in conjunction with tourniquet occlusion at aproximal site, resulted in uniform transduction of muscle fibers at anefficiency approaching 100%. Histochemical assays for Xgal activityrevealed levels an average 1000-fold higher than background in thelatter groups, while there was no detectable difference from backgroundin the rats receiving simple intravenous infusions of vector. Ratsinfused at lower pressure (50 torr) below a tourniquet had low but stilldetectable levels of transduction in an inhomogeneous pattern localizedprimarily near the distal site of infusion. Finally, anesthetized ratsin which a tourniquet was placed to encircle the caudal abdomen,(occluding the anatomic homologue to the human infrarenal aorta andinferior vena cava) showed homogenous transduction of musclessurrounding the pelvic girdle (e.g., gluteus). All animals tolerated theprocedures well, and gradually mobilized the interstitial fluid loadswithout signs of cardiopulmonary compromise as they rapidly returned totheir pre-procedural weights. Normalized to the titers of input vector,the absolute values of tissue X-gal were similar in animals infused withAAV vectors bearing capsids of serological classes 1 and 2.

Example 4 Canine Animal Model

As proportionally larger quantities of vector became available, similarstudies were undertaken in a large animal model. The results werescale-independent in comparisons between the rat and dog.

A. Intravascular Injection of rAd and rAAV in Dog Isolated Limbs

Mixed hound male canines 5-11 weeks in age and weighing 5-11 kg wereinfused with rAAV (10¹⁴° C.) and rAd (10¹³ particles) diluted in 500 mlof sterile PBS at a delivery pressure of 300 torr. Following intravenoussedation with medetomidinine hydrochloride (Dormitor® brand, NovartisAnimal Health) and butorphenol, dogs underwent greater saphenous veincannulation through a small incision (˜2 cm) with a 20 gaugeangiocatheter. An atraumatic tourniquet was placed at the level of thegroin, secured in place to prevent distal migration, and tightened untilthe femoral pulse could no longer be palpated. The infusion was begunwith the aid of a pressure bag inflated to the maximum level (300 torr)through standard IV tubing with care to ensure that no air could enterthe vein. The total time of infusion with dwell was twenty minutes. Thetourniquet was then released and the cannula was removed. The saphenousvein was ligated with a silk suture and the incision was closed withresorbable suture. Necropsies were performed at day 14. The sameprocedure was performed without tourniquet placement as control.

B. Results

Despite the readily discernable increase in the histological thicknessof the canine perimysial and epimysial fascia, the pattern of andextraordinary efficiency of transduction reflects uniform vectordistribution and extravasation throughout the entire hindlimb. Similarhistochemical results were obtained with adenovirus and AAV. Based onour earlier studies comparing X-gal staining for beta-galactosidase andimmunofluorescence staining for disease-specific transgene products, weexpected the current approach to meet the gene delivery requirements fortherapeutic efficacy in hemophilia and muscular dystrophy. All dogstolerated this procedure well, generally returning to full, symmetricalweight bearing within minutes of completion of the procedure.Periprocedural monitoring documented no significant alterations inpulse, blood pressure, or arterial oxygen saturation. Of note, theseprocedures were all performed using mild, rapidly reversible sedationwith medetomidinine hydrochloride (Dormitor® brand, Novartis AnimalHealth) and atipamezde hydrochloride (a medetomidinine reversing agent,Antisedan® brand. Novartis Animal Health), without the need forendotracheal intubation or mechanical ventilation. None of the dogs inthis series demonstrated any clinical signs of muscle or cardiovasculardysfunction referable to the procedure.

The experimental findings are consistent with the inventors' theory formechanical distention and perturbation of the endothelial sheet, and forthe pattern of generally afferent but locally retrograde flow throughthe venous arcade.

In mammals, most of the ultrastructural dimensions of themicrovasculature are scale-independent. A notable exception of relevanceto translational studies in gene therapy is the thickness or width ofthe basement membrane, a parameter which increases in proportion to theaverage transmural distending pressure. In all but the smallest mammals,constraints on regulation of the central venous pressure require thickerbasement membranes in the limbs than in the central circulation, perhapsexplaining the relative ease with which systemic gene delivery can beobtained in the mouse.

All documents identified herein are incorporated by reference. Numerousmodifications to, and variations of, the specific embodiments describedherein will be readily apparent to one of skill in the art. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1. A method for transferring a macromolecular complex to muscle cells ina subject, said method comprising the steps of exsanguinating a regionof the subject's microvasculature; delivering a macromolecular complexto the exsanguinated region of the subject's microvasculature under highhydrostatic pressure that transfers the complex to the muscle cells. 2.The method according to claim 1, further comprising the step ofisolating the subject's microvasculature in a limb prior toexsanguination of the limb.
 3. The method according to claim 1, whereinthe macromolecular complex is delivered systemically to the venous sideof the subject's microvasculature.
 4. The method according to claim 1,wherein the macromolecular complex is a viral vector carrying a targetmolecule.
 5. The method according to claim 1, wherein the targetmolecule is selected from the group consisting of an RNA molecule, atransgene, and a chemical moiety.
 6. The method according to claim 1,wherein the transgene is selected from the group consisting of FactorVIII, Factor IX, erythropoietin, a Muscular Dystrophy protein, and acardiomyopathy protein.
 7. The method according to claim 6, wherein theMuscular Dystrophy protein is selected from the group consisting ofdystrophin, utrophin, a sarcoglycan protein, calpain, Fukutin,Fukutin-related protein, telethonin, and laminin.
 8. The methodaccording to claim 1, wherein the hydrostatic high pressure is in therange of about 100 to 500 mm Hg.
 9. The method according to claim 1,wherein the hydrostatic high pressure is rapidly applied by way of atleast one low resistance catheter or cannula in either a vein or anartery.
 10. A method of administering a macromolecular complex to asubject having circulating antibodies to said macromolecular complex,said method comprising the steps of: isolating a region of the subject'smicrovasculature and exsanguinating the region; contacting, under highhydrostatic pressure, a subject's microvasculature with a solutioncomprising a macromolecular complex for delivery to the subject andsaline.
 11. The method according to claim 10, further comprising thesteps of flushing out residual macromolecular complex and resanguinatingthe patient.
 12. A method of administering a macromolecular complex tothe interstitial space of a subject without activating destructiveclotting factors or inflammatory response, said method comprising thesteps of: isolating a region of the subject's microvasculatureexsanguinating the region; contacting, under high hydrostatic pressure,a subject's microvasculature with a solution comprising a macromolecularcomplex for delivery to the subject and saline.
 13. A method oftransferring a macromolecular complex to a limb of a subject, saidmethod comprising the step of: (a) placing a proximal inflatabletourniquet or balloon catheter for isolating the vasculature of a limbof a subject; (b) exsanguinating the limb; (c) applying pressuresufficient to isolate the limb at a girdle between the limb and thetrunk of the subject's body; and (d) infusing the macromolecular complexinto the limb at a high hydrostatic pressure.
 14. The method accordingto claim 13, wherein the macromolecular complex is infused inoxygenated, physiologic saline for a total volume in the range of 20 to100% of the estimated volume of the extremity.
 15. A method ofsystemically transferring a macromolecular complex to muscle cells of asubject, said method comprising the step of: placing a patient undertotal circulatory arrest using a first heart-lung cannulae and a secondheart-lung cannula, each said cannula being placed in a suitable vesselthrough a cannulation site in each vessel; lowering the patient'stemperature to 15 to 18° C.; partially exsanguinating and decannulatingthe patient from the first and second heart-lung cannulae; introducing afirst balloon catheter and a second balloon catheter in the cannulationsites, wherein upon inflation the first balloon catheters occludes theaorta and the second balloon catheter occludes the venae cavae topreclude backflow; inflating the balloon catheters to a pressureexceeding that applied from cannulae in extremities of the patient; andsimultaneously applying the macromolecular complex in solution to allfour of the patient's extremities via the cannulae located therein. 16.The method according to claim 15, wherein the solution is allowed todwell for a period of about 5 to 30 minutes.
 17. The method according toclaim 15, further comprising the steps of: flushing out residualmacromolecular complex; withdrawing the balloon catheters; reinsertingthe heart-lung cannulae, resanguinating and rewarming the patient. 18.The method according to claim 15, wherein the balloon catheters comprisean inflatable balloon which extends the length of the cannulae such thatupon inflation it expands radially to abut the walls of the vessel, theaortic space and the venae cavae.
 19. (canceled)
 20. (canceled)
 21. Aballoon catheter for use in infusion of macromolecular complexes intothe venous microvasculature of a hypothermic patient, said ballooncatheter comprising: an inflatable balloon (111) having an interior,said balloon being expandable radially without significant distalexpansion; a flexible cannula (125) having a distal end and a proximalend and extending along an axis having a internal channel for thecontrolled application of fluid under pressure; wherein said balloon(111) is attached to the cannula at least two points, one point ofattachment being adjacent to the distal end of the cannula and a secondpoint of attachment being adjacent to the proximal end of the cannula,such that when inflated, said balloon expands radially to abut the wallsof a vessel in which it has been inserted and occludes the aortic spaceor the venae cavae.
 22. The balloon catheter according to claim 21,wherein the balloon (111) is attached at three or more locations alongthe cannula.
 23. The balloon catheter according to claim 21, wherein theballoon comprises at least two compartments (142, 144, 146) formed bymultiple points of attachment.
 24. The balloon catheter according toclaim 23 in which the balloon comprises at least three compartments(142, 144, 146) formed by multiple points of attachment, in which one ofsaid compartments (144) is an intermediate compartment situated alongthe length of the cannula between two other balloon compartments (142,146), and in which the distensibility of the intermediate compartment(144) is substantially lower than the distensibility of each of said twoother balloon compartments, whereby, when the balloon catheter isdisposed in the vena cavae of a patient, with said intermediatecompartment (144) situated in the right atrium of the patient's heart,expansion of said intermediate balloon compartment is prevented fromdistending the patient's heart excessively. 25-30. (canceled)
 31. A kitcomprising a balloon catheter and instructions for performing a methodaccording to claim
 1. 32. A kit comprising a macromolecular complex andinstructions for performing a method according to claim
 1. 33. A kitcomprising a balloon catheter according to claim 21 and instructions foruse thereof.